Chapter 5: Nucleic Acid Extraction

Deoxyribonucleic acid (DNA)

A linear polynucleotide consisting of four types of monomeric nucleotides.

Ribonucleic Acid (RNA)

A linear molecule containing four types of nucleotides linked by phosphodiester bonds

Endogenous Ribonucleases

Represent the major factor causing RNA degradation

RNA Integrity Number (RIN)

A software algorithm that takes the electrophoretic RNA measurements into account in order to assign integrity values to RNA samples

deoxyribose

The ______ is attached to the nitrogen of a base.

phosphate group

The ______ is attached to the deoxyribose.

phosphodiester

In a polynucleotide, individual nucleotides are linked by _____ bonds.

Friedrich Mescher

A Swiss physician to first attempt and accomplish isolate DNA from humans, which involved the use of human leucocytes.

nuclein

DNA molecule

enzymatic digestions

In most DNA extraction protocols, _____ are used for cell and tissue disruption.

disruption process

The ______ can also be carried out by boiling and by using alkali treatment and mechanical methods.

decalcification process

A _______ removes calcium ions from the matrix, thus making the specimen suitable for DNA extraction.

membranes

During or after tissue disruption, ______ are lysed in order to release DNAs.

DNases

A type of nuclease that catalyzes the cleavage of phosphodiester bonds of DNA.

Endogenous DNases

Are located in cytoplasmic lysosomes and play a role in degrading the DNA of invading viruses. When cells are lysed, the DNases that are also released can degrade the extracted DNA.

Chelating agents

Are used to chelate the divalent cations that are the cofactors of DNases, in order to inhibit DNase activities.

TE buffer

Purified high-molecular-weight DNA is usually stored in ______.

thawing cycles

Frequent freezing or ______ should be avoided because temperature fluctua- tions may cause breaks of single- and double-stranded DNA.

heavy metals

The presence of ______ can cause breakage of phosphodiester bonds in the molecules.

Contamination

It can occur between samples, between an individual and a sample, between other organisms and a sample, or between amplified DNA and a sample.

Cell Lysis and Protein Digestion

These steps can be achieved by digestion with proteolytic enzymes such as proteinase K before extraction with organic solvents.

Extraction with Organic Solvents

The removal of proteins is carried out by extracting aqueous solutions containing DNA with a mixture of phenol:chloroform:isoamyl alcohol.

Phenol

Is used to extract the proteins from the aqueous solution.

Chloroform

is utilized as it has a higher density than phenol

Isoamyl alcohol

is often added to the phenol–chloroform mixture to reduce foaming.

Ethanol Precipitation

The DNA is precipitated from the aqueous solution with ethanol and salts.

Ultrafiltration

An alternative to ethanol precipitation for concentrating DNA solutions.

Washing

This step removes contaminants and inhibitors that may interfere with DNA amplification.

Boiling

Cells are suspended in a solution and incubated at 56°C, where DNases are not active, for 20 min.

Centrifugation

This method is simple and rapid and uses only a single tube for extraction, thus reducing the risks of contamination and sample mix-ups.

Brief centrifugation

is performed to pull the Chelex® 100 resin and cellular debris to the bottom of the tube. The supernatant is used for DNA analysis.

Silica-Based Extraction

The method is based on the phenomenon that DNA is reversibly adsorbed to silica— silicon dioxide (SiO2)—in the presence of high concentrations of chaotropic salts.

Chaotropic salts

can disrupt hydrogen bonding, affecting the three-dimensional structures of macromolecules. These salts are used to denature proteins.

guanidinium salts

Common chaotropic salts utilized for DNA extraction include _____.

Cell Lysis and Protein Digestion

This is carried out by proteinase K digestion. The cell membranes are broken open, and DNA is released.

DNA Adsorption onto Silica

This step utilizes silica as the stationary phase in a membrane configuration to which the DNA in the cell lysate binds.

Washing

This step removes chaotropic agents and other contaminants. An ethanol-based wash solution is used.

Elution of DNA

The adsorbed DNA can be eluted by rehydration with aqueous low-salt solutions.

Differential Extraction

This method is very useful for the extraction of DNA from biological evidence derived from sexual assault cases, such as vaginal swabs and bodily fluid stains.

ribose

The sugar residue of RNA is a ______, which has a hydroxyl (OH) group at the 2′ carbon position.

messenger RNA

The RNA that carries codes from a DNA template is called ______ (mRNA) and usually contains a cap and polyadenine tail at the 5′ and 3′ ends of the molecule.

Endogenous Ribonucleases

Represent the major factor causing RNA degradation.

lysis buffer

The _______ usually contains chaotropic salts that can facilitate the lysis of cells and denature proteins, thus inactivating endogenous RNases to protect RNA.

high-salt environment

A _____ in the lysate allows selective binding of silica to genomic DNA over RNA.

miRNAs

Are low-molecular-weight RNAs ranging between 15 and 30 nucleotides in length.

organic-solvent extraction

It is used to isolate total RNA.

solid-phase extraction

It is used to enrich small RNA.

Single-use aliquots

Are preferred when possible to avoid multiple freeze–thaw cycles.